The chemokines are small polypeptides, generally about 70-100 amino acids (aa) in length, 8-11 kD in molecular weight, and active over a 1-100 ng/ml concentration range. Initially, chemokines were isolated and purified from inflamed tissues and characterized relative to their bioactivity. More recently, chemokines have been discovered through molecular cloning techniques and characterized by structural as well as functional analysis. Closely related polypeptides currently assigned to the .beta. chemokine family also display definitive spacing of the first two cysteine residues in the mature molecule and act on a diverse group of target cells, many of which are leukocytes and which include monocytes and macrophages, basophils, eosinophils, and T lymphocytes. The known chemokines and their functions are reviewed by Schall T J (1994; Chemotactic Cytokines: Targets for Therapeutic Development, International Business Communications, Southborough Mass., pp 180-270) and by Paul W E (1993; Fundamental Immunology, Raven Press, New York N.Y., pp 822-26).
Relatively few C--C chemokines have been fully described, but they are clearly distinct from the .alpha. or C--X--C chemokines and the .gamma. or C chemokines. Although both .alpha. and .beta. chemokines generally function as dimers, the .beta. chemokines appear to have less N-terminal processing. Known C--C chemokines include the macrophage chemotactic proteins (MCP), macrophage inflammatory proteins (MIP), I-309, TCA3, and RANTES.
Monocyte chemotactic protein (MCP-1) is a 76 amino acid mature protein which appears to be expressed in almost all cells and tissues upon stimulation by a variety of agents. According to Charo I, (personal communication), the targets of MCP-1 may be limited to monocytes and basophils in which it induces an mcp-1 receptor:G protein-linked calcium flux. Two other related proteins (MCP-2 and MCP-3) have been purified from a human osteosarcoma cell line. MCP-2 and MCP-3 have 62% and 73% amino acid identity, respectively, with MCP-1 and both share MCP-1's chemoattractant specificity for monocytes.
MIP-1.alpha. and MIP-1.beta. were first purified from a stimulated mouse macrophage cell line and elicited an inflammatory response when injected into normal tissues. At least three distinct and non-allelic genes encode human MIP-1.alpha., and seven genes, MIP-1.beta.. MIP-1.alpha. and MIP-1.beta. consist of 68-69 amino acids which are about 70% identical in their acidic, mature secreted forms. They are both expressed in stimulated leukocytes, particularly T cells, B cells and monocytes in response to mitogens, anti-CD3 and endotoxin, and both polypeptides bind heparin. While both molecules stimulate monocytes, MIP-1.alpha. chemoattracts the CD-8 subset of T lymphocytes and eosinophils, while MIP-1.beta. chemoattracts the CD-4 subset of T lymphocytes. In mouse, these proteins are known to stimulate myelopoiesis.
I-309 was cloned from a human .gamma.-.delta. T cell line and shows 42% amino acid identity to T cell activation gene 3 (TCA3) cloned from mouse. There is considerable nucleotide homology between the 5' flanking regions of these two proteins, and they share an extra pair of cysteine residues not found in other chemokines. Such similarities suggest I-309 and TCA3 are species homologs which have diverged over time in both sequence and function.
RANTES is a C--C chemokine with structural similarity to the interleukin-8 (IL-8) and human MIP-1.beta. (Covell D G et al. (1994) Protein Science 3:2064-72). It is expressed by T lymphocytes and macrophages and has been identified in some tumor cell lines and in rheumatoid synovial fibroblasts. Both human and murine forms of the 68 amino acid mature protein are known; they share 85% homology and some cross reactivity. The human gene is located on chromosome 17q11-q21.
RANTES expression can be stimulated using LPS, and it is regulated by interleukins-1 and -4, transforming nerve factor and interferon-.gamma.. The cDNA cloned from T cells encodes a basic 8 kD protein which lacks N-linked glycosylation and may or may not have O-linked glycosylation.
RANTES functions through the mobilization of calcium ions (Ca.sup.++ ; Meurer R et al. (1993) J Exp Med 178:1913-1921). When the heterotrimeric G-protein coupled receptors are involved, chemotaxis of monocytes, memory helper T cells (but not B cells), and eosinophils occurs. When protein tyrosine kinases are involved, cellular responses such as channel opening, interleukin 2 receptor expression, cytokine release and T cell proliferation occur (Bacon K B et al. (1995) Science 269:1727-29). RANTES has a different receptor than IL-8 and MIP-1.alpha.; however, it can displace MIP-1.alpha. from its normal receptor (Callard R and A Gearing (1994) The Cytokine Facts Book, Academic Press, San Diego Calif.).
RANTES also triggers the release of histamine from basophils and mast cells and activates T cells in an antigen-independent manner. Within four hours of intradermal injection in dog; RANTES, IL-8, and MCP-1 resulted in eosinophil and macrophage rich inflammation, neutrophil infiltration, and perivascular cuffing of monocytes, respectively.
The discovery of the novel RANTES homolog disclosed herein presents opportunities to intercede in inflammation, allergies, and asthma attributable to chemotaxis and other cellular responses mediated by such .beta. chemokines.